University of Groningen Bacillus subtilis: sporulation ... · Together, our data provide a unique...
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University of Groningen
Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNABoonstra, Mirjam
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Chapter 3
Analyses of the competent and
non-competent subpopulations
of B. subtilis reveal yhfW, yhxC
and ncRNAs as novel players in
competence.
Mirjam Boonstra1, Marc Schaffer2, Ulrike Mäder2, Petra Hildebrandt2,
Praveen Kumar Sappa2, Anne de Jong1, Joana Sousa3, Michael Lalk3, Uwe
Völker2, Oscar P. Kuipers1*
1. Molecular genetics group, Groningen Biomolecular Sciences and
Biotechnology Institute (GBB) University of Groningen;
2. Department of Functional Genomic, Interfaculty Institute of Genetics
and Functional Genomics, Ernst-Moritz-Arndt University, Greifswald,
Germany
3. Department of Cellular Biochemistry/ Metabolomics, Institute of
Biochemistry, Ernst-Moritz-Arndt University, Greifswald, Germany
Chapter 3
79
Abstract
One of the intriguing features of Bacillus subtilis is its ability to become
naturally competent and take up exogenous DNA. Under competence
stimulating conditions only part of population becomes competent. As yet
little is known about differential expression of non-coding RNAs and
differential protein levels of the competent and non competent
subpopulations. It is also not entirely clear if ComK is capable of direct
down regulation of genes or if it is solely a transcriptional activator. To
address these questions, we separated the competent and non-competent
subpopulations by use of FACS and used transcriptome sequencing (RNA-
seq) and proteome analysis (ESI-MS) to compare gene expression and
protein levels within the subpopulations. We found significant up-
regulation of expression of non-coding RNAs during competence, many of
which contain ComK binding sites (K-boxes) in their promoter region.
Thus, antisense ncRNAs play a role in down-regulation of specific genes
during competence. By investigating the proteome during competence we
found that the important competence factors MinD, Noc, SbcC and SbcD
are regulated at the post-transcriptional level. We also identified yhfW as a
gene the absence of which leads to a decrease in the expression of comG.
We found that yhfW has a modest but statistically significant effect on
comK expression, and also changes expression of another important
regulator gene, srfA. Metabolomic analysis of ΔyhfW under competence
stimulating conditions reveals significant reductions in TCA cycle
components and several amino acids. Interestingly in the ΔyhfW strain
there is higher expression of the NAD synthesis genes nadA, nadB and
nadC. Absence of YhfW causes a significant increase in the expression of
Spo0A under sporulation conditions. Inactivation of the neighbouring gene
of yhfW, yhxC decreases the total number of competent cells
approximately by half. Inactivation of yhxC also effects the expression of
comK and srfA. Together, our data provide a unique high resolution view
of competence development in B. subtilis and reveal a number of novel
factors involved in this process.
Chapter 3
80
Introduction
Bacillus subtilis is a Gram positive soil bacterium capable of developing
natural competence. During the competence state it can take up exogenous
DNA from the environment. Competent bacteria no longer undergo cell
division and are inhibited in their ability to synthesize new DNA (Briley Jr
et al., 2011; Hahn et al., 2015; Haijema et al., 2001; Mirouze et al., 2015).
Under nutrient limited conditions in the lab about 5-25 % of a B. subtilis
168 population becomes competent. The genes involved in competence are
under control of the master regulator of competence, ComK. The
competence state (K-state) of B. subtilis has previously been studied with
microarray techniques (Berka et al., 2002; Hamoen et al., 2002; Ogura et
al., 2002) and LacZ fusions (Ogura et al., 2002). To overcome the problem
posed by the small fraction of competent cells, these studies compared
comK and/or mecA deletion mutants with WT strains. In these studies
comK was found to be a transcriptional activator with only a few potential,
but not significantly, down-regulated genes found.
Despite the thorough investigations done in previous research, several
questions still remain unanswered, e.g.: Is there down-regulation of genes
in the competent subpopulation? Is there differential expression of non-
coding RNAs during competence? Are there competence proteins for which
regulation takes place primarily at the post transcriptional level?Here we
use deep RNA sequencing, proteomics and metabolomics to address these
questions. In contrast to previous studies we physically separated the two
subpopulations using a competence specific GFP reporter (PcomG-gfp) in
which the competent subpopulation expressing PcomG will be fluorescent
(Smits et al., 2005). We quickly fixed the cells using sodium chloride to
prevent degradation of RNA (Brown and Smith, 2009; Nilsson et al., 2014)
and used FACS to separate the competent and non competent
subpopulations. Separating and comparing the two subpopulations allowed
us to study a more natural situation than is created when using knock-outs
and allowed us to better determine whether significant down-regulation
occurs in the competent sub-population.
Competence subpopulations
81
Because of the higher sensitivity of RNA-seq compared to microarray
analyses we could also gain a better insight in operon expression, sRNA
occurrences and changes in weakly expressed genes that may not be
detected by standard microarray technology. Recently, the expression and
function of non-coding RNAs in B. subtilis has gained keen interest. Strain
168 is predicted to harbour approximately 100 potential small RNAs (Irnov
et al., 2010). More non-coding RNAs have been found in the condition-
depended transcriptome study by Nicolas (Nicolas et al., 2012).
However, little is known about the expression of ncRNAs in B. subtilis
during competence and we therefore decided to examine if differential
expression of non-coding RNAs occurs during competence. In the previous
gene expression studies certain genes known to be involved in competence
were not found differentially expressed at the RNA level. We therefore
decided to also investigate protein levels between the two subpopulations
in order to determine if regulation of some of the competence factors
occurs primarily on a post-transcriptional level.
Under the conditions used by us, B. subtilis becomes competent after
approximately 5 hours and transformability is highest during a two hour
window after entrance into the competent state. In this time window we
investigated changes in expression of the competent subpopulation and the
non-competent subpopulations. We discovered several new genes
differentially expressed during competence in our study, as well as ncRNAs
responsible for down regulation of important genes, and we decided to
investigate the role in competence of the most highly up regulated new
gene yhfW and its neighbouring gene yhxC in more detail.
Results
Differential expression of protein encoding genes
To gain insight into the progression of the competence state, B. subtilis 168
pcomG-gfp was grown in competence medium and cells were sorted by
FACS. Samples were taken early in the competence state at 5.5hrs and at a
later stage at 6.5hrs in order to gain insight in the progression of
competence.
Chapter 3
82
We subsequently compared the transcriptome and proteome of the
competent subpopulation with those of the non-competent subpopulation
at both time points.
To prevent degradation of RNA the cells were fixed with 2M NaCl in PBS
before FACS and sorted in to 4M NaCl in PBS (Brown and Smith, 2009;
Nilsson et al., 2014). The NaCl fixation method was tested by microarray
and RNA was isolated as described in (Nicolas et al., 2012). To exclude a
difference in sporulation initiation under these conditions, we looked
specifically at expression of sporulation genes. We did not find a significant
difference between the two subpopulations with respect to entry into
sporulation. After transcriptome analysis of the two subpopulations by T-
REx (de Jong et al., 2015) a total of 135 genes were found differentially
expressed between the competent and non-competent subpopulations at
5.5 hours (Table 1a) and 106 genes at 6.5 hours when using a cut off value
of 2-fold and an adjusted P-value of 0.05. The fold-difference in gene
expression at the RNA level was higher at the early time point, which was
expected since the changes in gene expression would be greatest upon
switching from one state to another. Our results were in accordance with
previous studies with regard to the core ComK regulon. Some of the genes
found previously were not found in our results. This was in part expected
due to the use of different strains of B. subtilis, different media and
possibly because of the use of knock out mutants used in previous studies.
Because of the high sensitivity of RNA-seq we also found several extremely
high fold change genes with a very low expression. We did not investigate
these genes further. In total we found 44 new genes for the first time point
and 38 in the second. Newly found highly up-regulated genes in the first
time point (>5fold) are yhfW, phrH, ygaK, ccpB, yvqJ, rsoA, ydeB, clpE,
maa, ybzI, sacB and yeeI. Some of these genes such as phrH, ccpB, maa
and ybzI are part of operons also found in previous studies. As in previous
studies we found a large number of genes of unknown function as well as
genes involved in metabolism. When only the protein coding genes are
used in comparing the competent and non-competent subpopulations a
total of 12 known regulatory genes were found differentially expressed.
Competence subpopulations
83
For nine of these we also found other genes in their regulon up-regulated.
Only for mta, ftsR and licT no other genes in their regulon were
differentially expressed. Unlike previous studies, we found also
significantly down-regulated genes, primarily at the first time point with
jag being the only gene down regulated at both time points. Two of the
down-regulated genes, i.e. ywdK and degS have not been previously
identified. The detection of degS was likely due to the sensitivity of RNA-
seq as the other gene in the operon degU has been previously detected.
Four of the down-regulated genes in this study were found up-regulated by
Berka et al. and two by Hamoen et al. These are degU, sigA, jag and lipL
(ywfL). None of these genes contain a K-box in the promoter region.
Deletion of jag, the only gene found down-regulated at both time-points,
did not result in a change in competence.
gene fold description previous
ssbB 199.3 single-stranded DNA-binding protein SsbB B,H,O
comGG 158.2 ComG operon protein 7 B,H,O
comEA 156.6 ComE operon protein 1 B,H,O
comGD 147.0 ComG operon protein 4 B,H,O
comC 139.0 type 4 prepilin-like proteins leader peptide-processing enzyme B,H,O
comGA 129.7 ComG operon protein 1 B,H,O
comGE 129.2 ComG operon protein 5 B,H,O
comGF 119.5 ComG operon protein 6 B,H,O
comGB 119.1 ComG operon protein 2 B,H,O
comFA 116.2 ComF operon protein 1 B,H,O
comGC 101.4 ComG operon protein 3 B,H,O
yqzE 90.6 hypothetical protein B,H
nucA 73.1 DNA-entry nuclease B,H,O
comFB 63.2 ComF operon protein 2 B,H,O
comFC 58.1 ComF operon protein 3 B,H,O
ybdK 56.3 sensor histidine kinase B,H,O
yhfW 52.5 rieske 2Fe-2S iron-sulfur protein YhfW
rapH 51.9 response regulator aspartate phosphatase H B,H,O
nin 51.9 DNA-entry nuclease inhibitor B,H,O
comEB 51.2 ComE operon protein 2 B,H,O
phrH 48.0 inhibitor of regulatory cascade
Table 1. Differential expression of protein encoding genes at the first time point, after 5.5hrs growth in competence medium. The last column indicates if the gene was found differentially expressed in the previous transcriptomics studies B: Berka, H: Hamoen, O: Ogura (Berka et al., 2002; Hamoen et al., 2002; Ogura et al., 2002). Table continues on following pages
Chapter 3
84
gene fold description previous
comEC 47.8 ComE operon protein 3 B,H,O
sacX 46.4 negative regulator of SacY activity B,H,O
radC 45.3 hypothetical protein B,H,O
yyaF 44.4 GTP-dependent nucleic acid-binding protein EngD B,H,O
maf 42.7 septum formation protein Maf B,H,O
exoAA 39.9 exodeoxyribonuclease B,H,O
comK 39.4 competence transcription factor B,H,O
dprA 31.9 protein smf B,H,O
yvyF 30.9 hypothetical protein B,H,O
hxlR 30.0 HxlR family transcriptional regulator B,H
spoIIB 29.1 stage II sporulation protein B H
sacY 24.9 levansucrase and sucrase synthesis operon antiterminator H,O
coiA 23.9 competence protein CoiA H,B
ygaK 20.4 FAD-linked oxidoreductase YgaK
rpsF 18.2 30S ribosomal protein S6 O
yhcE 17.7 hypothetical protein B
yviE 15.9 hypothetical protein B
ssbA 15.1 single-stranded DNA-binding protein B
yhcF 14.0 GntR family transcriptional regulator H,O
rpsR 13.8 30S ribosomal protein S18 B
yvrO 13.8 ABC transporter ATP-binding protein B
recA 13.8 recombinase RecA B,H,O
yckB 12.0 ABC transporter extracellular-binding protein YckB B,H
glcR 11.6 HTH-type transcriptional repressor GlcR H,O
ccpB 11.5 catabolite control protein B
yckA 11.4 amino acid ABC transporter permease protein YckA B,H
ycbP 11.0 hypothetical protein B,H
hxlB 10.9 3-hexulose-6-phosphate isomerase B,H
guaD 10.1 guanine deaminase H
yvqJ 10.0 MFS transporter
hxlA 9.9 3-hexulose-6-phosphate synthase B,H
yviF 9.6 flagellar assembly factor FliW B
groES 9.4 10 kDa chaperonin B,H
flgM 9.3 negative regulator of flagellin synthesis B,H
yvyG 9.2 hypothetical protein B,H
ydeE 9.1 AraC family transcriptional regulator B
ywpJ 9.0 phosphatase YwpJ B.H
rsoA 8.7 sigma-O factor regulatory protein RsoA
flgK 8.6 flagellar hook-associated protein 1 B
oxdC 8.5 oxalate decarboxylase OxdC B
csrA 8.4 carbon storage regulator homolog B
flgL 8.4 flagellar hook-associated protein 3 B
yhcG 8.1 spermidine/putrescine ABC transporter ATP-binding protein O
med 7.8 transcriptional activator protein med B,H
ydeB 7.6 transcription factor YdeB
Competence subpopulations
85
gene fold description previous
groEL 7.5 60 kDa chaperonin
yhcH 7.3 ABC transporter ATP-binding protein H,O
clpE 7.1 ATP-dependent Clp protease ATP-binding subunit ClpE
maa 7.0 maltose O-acetyltransferase
yvhJ 6.8 transcriptional regulator YvhJ B
trpB 6.8 tryptophan synthase beta chain B,H
yhcI 6.6 hypothetical protein B,H
yvyE 6.6 IMPACT family member YvyE B
yvrN 6.5 ABC transporter permease B
yvrP 6.2 efflux system protein YvrP B,H,O
sucC 6.2 succinyl-CoA ligase [ADP-forming] subunit beta H
ybzI 6.1 hypothetical protein
gid 6.0 TrmFO
sucD 5.8 succinyl-CoA ligase [ADP-forming] subunit alpha B,H
sacB 5.6 levansucrase
topA 5.6 DNA topoisomerase 1 B,H
comZ 5.5 ComG operon repressor B,H
yeeI 5.1 transcriptional regulator
ypzG 5.0 hypothetical protein
ybdJ 5.0 transcriptional regulator
spbC 4.7 killing factor SdpC
yjcM 4.4 hypothetical protein
yopL 4.4 hypothetical protein
ydzE 4.2 permease
radA 4.1 DNA repair protein RadA
ymzE/2 3.9 Pseudogene
yxiP 3.9 hypothetical protein H
holA 3.9 hypothetical protein
eglS 3.9 endoglucanase
sigO 3.9 RNA polymerase sigma factor SigO
yoqW 3.8 hypothetical protein
yomL 3.7 hypothetical protein B
yjiA 3.7 hypothetical protein
yuaG 3.6 hypothetical protein H
parA 3.6 sporulation initiation inhibitor protein Soj
mta 3.5 HTH-type transcriptional activator mta
yocI 3.4 ATP-dependent DNA helicase RecQ
ywtF 3.4 transcriptional regulator YwtF B
parB 3.4 stage 0 sporulation protein J
gidB 3.4 ribosomal RNA small subunit methyltransferase G B
ycgP 3.3 hypothetical protein
ytzJ 3.3 hypothetical protein
ftsR 3.2 LysR family transcriptional regulator
ywfI 3.2 heme peroxidase B,H
ykuK 3.1 hypothetical protein B,H
Chapter 3
86
gene fold description previous
bdbC 3.1 disulfide bond formation protein C B,H
dinB 3.0 protein DinB B,H
hrcA 2.8 heat-inducible transcription repressor HrcA
yeeK 2.8 spore coat protein YeeK
bdbD 2.8 disulfide bond formation protein D B,H
mcsA 2.7 hypothetical protein
licT 2.7 transcription antiterminator LicT
bpr 2.7 bacillopeptidase F
trmE 2.6 tRNA modification GTPase MnmE O
ywhH 2.5 hypothetical protein B
gidA 2.5 MnmG
mreB 2.4 rod shape-determining protein MreB B
yfhB 2.4 isomerase B
mcsB 2.4 ATP:guanido phosphotransferase YacI
yddT 2.3 hypothetical protein
yfhC 2.3 NAD(P)H nitroreductase YfhC B
comN 2.2 post-transcriptional regulator
aroD 2.2 3-dehydroquinate dehydratase
degS -2.3 signal transduction histidine-protein kinase/phosphatase DegS (B up)
sigA -2.4 RNA polymerase sigma factor RpoD
ywdK -2.5 hypothetical protein (B up)
DegU -2.7 transcriptional regulatory protein DegU (B up)
jag -2.8 protein jag (B,H up)
ywfL -3.0 octanoyl-[GcvH]:protein N-octanoyltransferase (B,H up)
Expression patterns of non-coding RNAs
Little is known about differential expression within the subpopulations of
non-coding RNAs during competence. We therefore decided to look at their
expression patterns. We found at T1 a total of 36 elements (47 including
high fold-change, but low expression) of which 17 are antisense RNAs
(Table 2) and at T2, 25 differentially expressed ncRNAs. The previously
found up-regulated genes degU, sigA, jag and lipL were found to have up-
regulated anti-sense RNAs instead of up-regulated mRNAs, which caused
the misinterpretation found in previous microarray analyses. The detection
of these genes as being up-regulated in the previous studies was likely the
result of the use of amplicon arrays in these studies. Because the probes are
made from double stranded (dsDNA) it cannot distinguish between sense
and antisense DNA.
Competence subpopulations
87
Hamoen et al. already determined that comER was one of these false
positives, and indeed we found up-regulation of the antisense comER RNA
(S963), but not of comER itself. Hamoen et al. also found up-regulation of
lipL and ywfM and Berka et al. up-regulation of lipL, ywfM, and pta; these
genes form an operon consisting of pta, cysl, lipL and ywfM. In fact Berka
et al. investigated expression of pta during competence using a promoter-
lacZ fusion, but could only see a small effect on expression during
competence, whereas one of their array comparisons suggested a robust
effect. As with comER this operon is covered by a large antisense RNA
called S1458, which we find up-regulated.
To determine whether expression of the ncRNAs could be controlled by
ComK we looked at the presence of potential K-boxes in their respective
promoter regions using genome2DTFBS. We found potential K-boxes for 7
of the ncRNAs within the first 100bp upstream region and 2 ncRNAs with
K-boxes within the first 300bps (Table 2). 10 ncRNAs are preceded by
competence genes with K-boxes in their respective promoter regions. The
majority of the antisense RNAs are preceded by potential K-boxes. We did
not find ncRNAs with a K-box at the second time point that were not
present at the first time point. Although we found 17 antisense RNAs, only
four of the up-regulated antisense RNAs have corresponding down-
regulated genes. Down-regulation of degU could be a direct result of the
up-regulation of its anti-sense RNA S1354 as DegU has been shown to
directly regulate its own expression (Ogura and Tsukahara, 2010).
The down-regulated lipL has been shown to be essential for biosynthesis of
lipoic acid, an enzyme co-factor (Christensen et al., 2011; Martin et al.,
2011). One of the most important enzyme complexes requiring lipoic acid is
the pyruvate dehydrogenase complex (Perham, 2000). Recently it has been
shown that the E1α subunit of this complex is important for correct Z ring
formation, and the complex is indicated as playing a role in the
coordination of nutrient availability and cell division (Monahan et al.,
2014). S1458, the antisense RNA to lipL, also contains a potential K-box in
the promoter region. S1458 is a very large antisense RNA covering four
genes (pta, cysl, lipL and ywfM). In Escherichia coli deletion of pta has
been found to rescue cell division in dnaA, dnaB, dnaE, dnaG and dnaN
mutants (Maciąg-Dorszyńska et al., 2012).
Chapter 3
88
S1579, i.e. the jag and spoIIIJ antisense RNA, was also up-regulated in our
data. Up-regulated S951 is antisense to sigA and partially overlaps dnaG.
The only down-regulated gene not covered by an antisense RNA was ywdK.
ywdK has previously been found to be negatively regulated by TnrA under
Nitrogen limiting conditions (Yoshida et al., 2003).
The presence of up-regulated anti-sense RNA and the down-regulation of
the genes confirm that the up-regulation of these genes found in the
previous studies were false positives of the same nature as found for
comER. Aside from the antisense RNAs there are also many 3'UTR and
5'UTR RNAs up-regulated. Some of these are at the 3’ or 5' end of up-
regulated genes, but others are in front of or behind genes that have no
known relation to competence. Interestingly, the competence gene noc that
has not been found to be differentially expressed in any of the
transcriptome studies is preceded by the ncRNA S1577, which is up-
regulated in the competent subpopulation.
name fold antisense description K-box
bp distance to start
transcript
S963 184.6 comER 5'UTR of comEA II-14 31
S962 173.6 yqzM independent transcript comE
S1354 167.8 degU independent transcript I-13 65
S1458 166.4 pta 5'UTR of hemQ I-15 29
S98 121.5 cwlJ 5'UTR of ycbP II-14 0
S122 117.4 bglC intergenic region nucA
S125 113.2 tlpC 5'UTR of hxlR II-13 95
S1399 100.8 3'UTR of ssbB ssbB
S652 98.1 yndK 3' of S653 no
S1579 96.6 spoIIIJ independent transcript II-15 5
S97 93 ycbO 3'UTR of ycbP no
S925 80.3 yqzG 3'UTR of yqzE comG
S245 43.4 intergenic region rapH
Table 2. Differential expression of ncRNAs at the first time point. The description is taken from Nicolas et al. 2012 The second last column indicates if the ncRNA has a K-box predicted by Genome2D TFBS. The type of K-box was manually determined according to the specifications used by Hamoen et al., 2002. The last column indicates the distance of the K-box to the start of the transcript, measured from the end of the K-box to the start codon.
Competence subpopulations
89
name fold antisense description K-box
bp distance to start
transcript
S1357 32.3 5'UTR of yvyE no
S1575 27.9 5'UTR of rpsF no
S401 26 yjzB intergenic region med
S1175 24.2 5'UTR of mntA II-15 51
S1353 22.3 intergenic region comF
S366 22.1 yhxD intergenic region comK
S655 21.5 yndL 5' of S653 no
S367 17.3 yhxD intergenic region comK
S951 16.1 sigA independent transcript no
S876 11.3 aroC 3'UTR of serA no
S1278 10.6 5'UTR of oxdC no
S583 10.2 5'UTR of topA I-13 275
S653 9.6 independent transcript no
S208 8.9 5'UTR of groES no
S209 8.3 3'UTR of groEL no
S967 5.8 3'UTR of sda no
S959 4.6 intergenic region no
S30 4 5'UTR of sspF no
S1577 3.2 intergenic region trmE 256
S174 3.1 3'UTR of yddM no
S515 2.8 intergenic region no
S296 -2.9 5'UTR of yfhP no
S488 -5.4 5'UTR of ykvA no
Differential protein levels between the competent and non-competent
subpopulations
B. subtilis 168 cells, sampled at 5.5 and 6.5hrs, were sorted by FACS onto a
filter manifold system. The filters were collected and stored at -80 0C.
Samples were digested and analysed by ESI-MS. At the first time point we
found 53 proteins to be differentially expressed, six of which were
downregulated in the competent subpopulation (Table 3). The second time
point had 94 differentially expressed proteins, 20 of which were down-
regulated in the competent fraction (Table 4)
Chapter 3
90
The higher number of proteins found at the second time point can be
explained by the maturation time of the proteins. 23 of the proteins found
in the first time point and 20 of the proteins found in the second time point
were also found in the RNA-seq data.
None of the genes found down-regulated in the RNA data were found
down-regulated at the protein level. This is likely caused by the shorter half
live of mRNA compared to that of proteins. None of the down-regulated
genes found in the protein data were found in the RNA-seq data. Most of
the down regulated proteins are involved in metabolism, with a few
unknown genes at the second time point.
As we expected, some of the proteins involved in competence that were not
found differentially expressed at the RNA level in our and the previous
studies, were found in the proteomics data. For some of these proteins the
gene is part of an operon, where other genes were differentially expressed
at the RNA level. The gene of cell division inhibitor MinD lies in an operon
with mreB, radC and maf. The gene noc is part of the trmE operon of
which thdF gidA and gidB were also upregulated on a RNA level. The genes
of SbcC, SbcD, however, do not reside in an operon of which the other
genes were differentially expressed at the RNA level. Ogura et al. found
differential expression of sbcC in one of their replicates, but none of the
other studies found differential expression of these genes. This indicates
that the regulation of SbcC and SbcD during competence takes place
primarily on a post-transcriptional level. The only known competence
genes that were not found differentially expressed in our study at either
RNA or protein level were addA, addB, hlpB, yhjB and yhjC. With the
exception of yhjB which has been found by Ogura et al, none of these genes
have been found significantly up-regulated in any of the four studies. This
strongly indicates that basal expression levels of these genes are sufficient
for competence.
Other interesting proteins with higher levels in the competent
subpopulation are the fatty acid biosynthesis proteins FabHA and FabF
(5.5hrs), and FloT which is involved in regulation of membrane fluidity.
Higher levels of these proteins may indicate a difference in fluidity and
lipid composition of the membrane during competence.
Competence subpopulations
91
In the same operon as the known competence gene coiA lies pepF, for
which we found higher protein levels in the competent sub-population.
Over-expression of pepF has been shown to inhibit sporulation initiation
(Kanamaru et al., 2002).
protein logFC description
ComEB 6.48 late competence protein required for DNA binding and uptake
NucA 6.24 catalyzes DNA cleavage during transformation
Nin 5.69 inhibitor of the DNA degrading activity of NucA
RecA 4.17 homologous recombination
SsbA 4.14 single-strand DNA-binding protein
YyaF 3.86 GTP-binding protein/ GTPase
FlgL 3.11 flagellar hook-associated protein 3 (HAP3)
FliW 2.78 checkpoint protein for hag expression, CsrA anatagonist
YdeE 2.64 similar to transcriptional regulator (AraC family)
YvrP 2.44 Unknown
TrmFO 2.35 tRNA:m(5)U-54 methyltransferase, glucose-inhibited division protein
Maa 1.96 maltose O-acetyltransferase
SucD 1.79 succinyl-CoA synthetase (alpha subunit)
SucC 1.70 succinyl-CoA synthetase (beta subunit)
YlbA 1.67 Unknown
FloT 1.59 involved in the control of membrane fluidity
TagT 1.57 phosphotransferase, attachment of anionic polymers to peptidoglycan
Noc 1.46 spatial regulator of cell division to protect the nucleoid
BdbD 1.41 required for the formation of thiol disulfide bonds in ComGC
Ffh 1.40 signal recognition particle (SRP) component
Spo0J 1.36 chromosome positioning near the pole, antagonist of Soj
SipW 1.25 signal peptidase I
GidA 1.24 glucose-inhibited division protein
ThdF 1.23 GTP-binding protein, putative tRNA modification GTPase
YckB 1.23 similar to amino acid ABC transporter (binding protein)
GrpE 1.21 heat-shock protein (activation of DnaK)
YfmM 1.17 similar to ABC transporter (ATP-binding protein)
YwfH 1.14 short chain reductase
SbcD 1.12 exonuclease SbcD homolog
MurB 1.10 UDP-N-acetylenolpyruvoylglucosamine reductase
Table 3. Difference in protein levels in the competent subpopulation at T1 Table continues on following pages
Chapter 3
92
protein logFC description
YdgI 1.05 similar to NADH dehydrogenase
YvbJ 1.01 Unknown
ClpY 1.01 two-component ATP-dependent protease, ATPase subunit
HemQ 0.99 heme-binding protein, essential for heme biosynthesis
FabHA 0.98 beta-ketoacyl-acyl carrier protein synthase III
ZosA 0.95 zinc transporter
HprT 0.93 hypoxanthine phosphoribosyltransferase
SwrC 0.91 similar to acriflavin resistance protein
GroEL 0.90 chaperonin and co-repressor for HrcA
FabF 0.89 Iinvolved in the control of membrane fluidity
YtsJ 0.83 malic enzyme
MinD 0.81 cell-division inhibitor (septum placement)
SbcC 0.79 DNA exonuclease
PepF 0.77 Oligoendopeptidase
DltC 0.76 D-alanine carrier protein
YtwF 0.70 Unknown
YqaP 0.68 Unknown
HisD -0.80 histidinol dehydrogenase
PyrAA -0.86 carbamoyl-phosphate synthetase (glutaminase subunit)
PheS -0.99 phenylalanyl-tRNA synthetase (alpha subunit)
HisG -1.12 ATP phosphoribosyltransferase
GudB -1.23 trigger enzyme: glutamate dehydrogenase
AtpF -0.83 ATP synthase (subunit b)
Competence subpopulations
93
protein logfc description
Nin 6.67 inhibitor of the DNA degrading activity of NucA
YyaF 4.45 GTP-binding protein/ GTPase
RecA 4.25 DNA repair/ recombination
FliW 3.16 checkpoint protein for hag expression, CsrA anatagonist
YvrP 3.06 unknown
ComGA 2.95 late competence gene, traffic ATPase
TrmFO 2.89 glucose-inhibited division protein
TopA 2.67 DNA topoisomerase I
RapA 2.40 dephosphorylates Spo0F-P, control of the phosphorelay
YwrO 2.35 similar to NAD(P)H oxidoreductase
Maa 2.31 maltose O-acetyltransferase
ThdF 2.10 GTP-binding protein, putative tRNA modification GTPase
Noc 2.06 spatial regulator of cell division to protect the nucleoid
Bcd 2.05 valine, isoleucine and L-leucine dehydrogenase
SucD 1.97 succinyl-CoA synthetase (alpha subunit)
TagV 1.97 attachment of anionic polymers to peptidoglycan
bdbD 1.94 required for the formation of thiol disulfide bonds in ComGC
SipW 1.87 signal peptidase I
FlgL 1.86 flagellar hook-associated protein 3 (HAP3)
YdeE 1.83 similar to transcriptional regulator (AraC family)
SucC 1.77 succinyl-CoA synthetase (beta subunit)
FabHA 1.75 beta-ketoacyl-acyl carrier protein synthase III
Spo0J 1.75 antagonist of Soj-dependent inhibition of sporulation initiation
InfC 1.70 translation initiation factor IF-3
SbcC 1.67 DNA exonuclease
Soj 1.63 negative regulation of sporulation initiation
RsmG 1.58 glucose-inhibited division protein
BacB 1.56 biosynthesis of the antibiotic bacilysin
YfhC 1.55 unknown
Ndk 1.50 nucleoside diphosphate kinase
YqhL 1.49 membrane protein
FloT 1.49 involved in the control of membrane fluidity
MetC 1.46 cystathionine beta-lyase
YpgR 1.45 unknown
YckB 1.43 similar to amino acid ABC transporter (binding protein)
Table 4. Difference in protein levels in the competent subpopulation at T2 Table continues on following pages
Chapter 3
94
protein logfc description
YydD 1.43 unknown
AnsB 1.38 L-aspartase
DltA 1.35 biosynthesis of teichoic acid
yjbL 1.34 unknown
GidA 1.31 glucose-inhibited division protein
YkuJ 1.29 unknown
YtxJ 1.28 general stress protein
SbcD 1.21 exonuclease SbcD homolog
YkvS 1.16 unknown
Ffh 1.13 signal recognition particle (SRP) component
McsB 1.11 modulator of CtsR-dependent repression
YvaK 1.10 general stress protein, similar to carboxylesterase
MurB 1.08 UDP-N-acetylenolpyruvoylglucosamine reductase
GroEL 1.08 chaperonin and co-repressor for HrcA
YeeI 1.07 unknown
HemQ 1.07 heme-binding protein, essential for heme biosynthesis
GroES 1.06 chaperonin, universally conserved protein
ClpY 0.99 two-component ATP-dependent protease, ATPase subunit
ThiC 0.95 biosynthesis of the pyrimidine moiety of thiamine
Rho 0.93 transcriptional termination protein
DapA 0.92 dihydrodipicolinate synthase
YkpA 0.90 similar to ABC transporter (ATP-binding protein)
EcsA 0.84 regulation of the secretion apparatus and of intra-membrane proteolysis
ClpC 0.83 ATP-dependent Clp protease, ATPase subunit
MinD 0.82 cell-division inhibitor (septum placement)
YvbJ 0.81 unknown
PepF 0.78 oligoendopeptidase, inhibits sporulation upon overexpression
HisS 0.77 histidyl-tRNA synthetase
YheA 0.76 unknown
YugI 0.72 unknown
YxkC 0.71 unknown
WalR 0.71 two-component response regulator, controls cell wall metabolism
AsnS 0.70 asparagyl-tRNA synthetase
DnaK 0.68 class I heat-shock protein (molecular chaperone)
YsaA 0.64 unknown
Zwf 0.62 glucose 6-phosphate dehydrogenase, pentose-phosphate pathway
Competence subpopulations
95
protein logfc description
YorD 0.62 unknown
YwfH 0.61 short chain reductase
LeuD -0.58 3-isopropylmalate dehydratase (small subunit)
MsrA -0.59 peptide methionine sulfoxide reductase
PheT -0.59 phenylalanyl-tRNA synthetase (beta subunit)
yxjH -0.66 putative methionine synthase
YqzC -0.71 unknown
AmhX -0.72 amidohydrolase
YpsC -0.75 similar to SAM-dependent 23S rRNA methyltransferase
YukE -0.82 unknown
YxiE -0.97 unknown
gpsA -1.07 glycerol-3-phosphate dehydrogenase (NAD)
YmcB -1.08 unknown
SdaAA -1.12 L-serine deaminase
HisF -1.23 cyclase-like protein
pyrB -1.29 aspartate carbamoyltransferase
MetQ -1.30 methionine ABC transporter (binding lipoprotein)
AlsS -1.42 acetolactate synthase
PckA -1.44 phosphoenolpyruvate carboxykinase
YvaQ -2.09 membrane-bound chemotaxis receptor
YeeB -2.23 unknown
GsaB -2.56 glutamate-1-semialdehyde aminotransferase
Differential expression within the subpopulations at T1 and T2 at RNA
and protein level
For both the competent and non-competent populations on the RNA level
as well as the protein level The difference was primarily in genes and
proteins involved in primary metabolism. More genes were significantly
changed in expression between the two time points in the competent
fraction compared to the non-competent fraction. At the first time point
there was a higher expression of aminoacid biosynthesis genes, in
particular arginine and histidine synthesis genes as well as increased
expression of phosphate uptake genes. Protein levels of arginine,
asparagine, leucine, methionine and threonine synthesis proteins were
more abundant at T1.
Chapter 3
96
Higher levels of amino-acid synthesis may be the result of increased
demand for the production of the large competence machinery. There were
also higher levels of proteins involved in the synthesis of purine and
pyrimidine in both subpopulations at the first time point. Not only were
there higher levels of amino acid synthesis proteins at the first time point,
the levels of RpsJ and RpsL, i.e. the ribosomal s10 and s12 proteins were
also higher. At T2 a significant down-regulation of groES and RNA binding
protein hfq in the competent subpopulation at the RNA level was found. In
the non-competent subpopulation there was also significant up-regulation
of rsoA, comGD, radC and ssbB and down-regulation of fliT at T2. On the
protein level at T2 the RNA polymerase beta subunit proteins RpoB and
RpoC had increased levels in both subpopulations. There were decreased
levels of SrfAA, SrfAC and SrfAD in both the competent and non-
competent subpopulations at T2 whereas levels of MecA were increased in
the second time point, which one might expect since competence is a
transient state requiring removal of ComK to leave it. With regards to
differential expression of ncRNAs between both time point there was very
little difference with only. S963 that is antisense to comER was the only
antisense RNA significantly changed in expression between the two time
points in the non-competent subpopulation.
Investigation into yhfW and yhxC
Among the newly found genes in competence, yhfW was the highest up-
regulated new gene. The only gene found down-regulated at both time
points is jag. Deletion of jag did not result in a significant change in
competence. As yhfW is up-regulated to a similar level as known
competence genes we hypothesised that yhfW is involved in competence
and that a deletion would lead to a reduction in competence. However,
deletion of the yhfW gene did not lead to a strong decrease in the amount
of competent cells, rather the expression of comG was significantly reduced
(Fig. 1A). To determine how YhfW might be affecting competence we
looked at the effect of inactivation of yhfW on the expression of known
competence regulators.
Competence subpopulations
97
We tested the expression of comK, srfA, spo0A and rok. In the mutant the
comK (Fig. 1B) expressing population was larger, but the intensity is
slightly reduced when maximum competence is achieved.
This difference was statistically significant before full formation of the
competent and non-competent sub-populations.
Fig.1. Differences in expression under competence stimulating conditions. Blue: control Red: BFA1698 (ΔyhfW) A: expression of PcomG-gfp . The difference is statistically significant , P <0.04-0.001. B: Expression of PcomK-gfp The difference is statistically significant at 3-4hrs P<0.001. C: Expression of PsrfA-gfp statistically significant at 2-3hrs P0.002-0.019. Statistics were performed using the Mann-Whitney Rank-Sum-test.
Chapter 3
98
Expression of srfA (Fig. 1C) was also significantly increased in the mutant
before on-set of competence and reduced later in the stationary phase,
although not statistically significantly so. Expression of spo0A was lower in
the mutant, but this effect is not statistically significant. There also was no
statistically significant effect on the expression of rok under competence
stimulating conditions.
Fig.2. Differences in expression of regulator expression under competence stimulating conditions. Blue: control Red: BFA1698 (ΔyhfW) A: Expression of Pspo0A-gfp the difference is not statistically significant. B: Expression of Prok-gfp the difference is not statistically significant. Statistics were performed using the Mann-Whitney Rank-Sum-test.
Competence subpopulations
99
The expression pattern of yhfW is nearly identical to that of its neighbour
yhxC, which is transcribed in the opposite direction (Nicolas et al., 2012).
yhfW has been shown to be regulated by SigF and yhxC by SigE
(Arrieta‐Ortiz et al., 2015; Wang et al., 2006). However there is not a
significant difference in sporulation regulators and their regulons between
the two subpopulations during competence, so we can exclude effects of
subpopulations going differently into sporulation. Both genes also share a
number of predicted regulator binding sites (Table 5). We therefore
decided to also investigate the effect of inactivation of yhxC on
competence. In the absence of yhxC the total amount of competent cells is
significantly reduced by approximately a factor of two (Fig3. A). In contrast
to ΔyhfW the expression of comK is reduced in the mutant, and again this
difference is only significant before maximum competence is achieved (Fig.
3B).As for ΔyhfW the expression of srfA is significantly increased in ΔyhxC
before the on-set of competence (Fig. 3C). The expression of spo0A is
slightly lower, but as for yhfW not statistically significant (Fig. 4A). As for
ΔyhfW, inactivation of yhxC does not significantly affect the expression of
rok (Fig. 4B). The transformability of the ΔyhfW strain is 1.4X lower than
the control, however this difference is not statistically significant. The
transformability of ΔyhxC is 2 times lower and is statistically significant.
predicted regulator yhfW yhxC
CcpC
CitT
CtsR
DegU
GltR
RocR
Xre
Zur
Table 5.Predicted sigma factor and regulator binding sites Regulator binding sites in the 300bp promoter region of yhfW and yhxC. All were predicted by Genome2D TFBS search.
Chapter 3
100
Fig. 3. Differences in expression under competence stimulating conditions. Blue: control Red: BFA1701 (ΔyhxC) A. expression of PcomG-gfp . The difference is statistically significant P <0.001 B. Expression of PcomK-gfp The difference is statistically significant at 2hrs P0.008 C. Expression of PsrfA-gfp statistically significant at 2 and 4hrs P<0.001-0.014 Statistics were performed using the Mann-Whitney Rank-Sum-test.
Competence subpopulations
101
Fig.4. Differences in expression under competence stimulating conditions. Blue: control Red: BFA1701 (ΔyhxC) A. Expression of P spo0A-gfp the difference is not statistically significant. B. Expression of Prok-gfp the difference is not statistically significant.
Statistics were performed using the Mann-Whitney Rank-Sum-test.
Chapter 3
102
Effect of yhfW inactivation on the metabolome
Both YhfW and YhxC are predicted oxidoreductases of unknown function.
YhfW is predicted to be a FAD-linked oxidoreductase and contains a Rieske
2Fe-2S domain at the C-terminus as indicated by Interpro. YhxC belongs to
the Short Chain Dehydrogenase (SDR_c1) family of proteins and shows
similarity to FabG and 3-oxo-ACP reductase domains (NCBI-pBLAST).
Proteins similar to YhfW and YhxC (Blast-P) are present in strains of many
species of Bacillus closely related to B. subtilis. Most of these Bacillus
species also contain ComK, Spo0A, and SrfA. Proteins with high similarity
to YhfW and YhxC are also found in Paenibacillus, Geobacillus,
Anoxybacillus, Fictibacillus, Brevibacillus species and several other
members of the Bacillaceae genus. Interestingly, when firmicutes were
excluded from the YhfW Blast, similar proteins are found in Archaea and
Cyanobacterial species, but not in Gram negative bacteria (100 species cut-
off), but in contrast YhxC like proteins are found in gram negative bacteria.
Because YhfW is predicted to be an enzyme we decided to determine if
inactivation of this gene has an effect on the metabolome under
competence conditions. A growth curve was determined to inspect possible
differences in growth between the mutant and the control, which are
shown to be nearly identical (S10). Samples were taken when maximum
comG-gfp expression was achieved, for this experiment 6-7 hrs. The
intracellular metabolome revealed differences in metabolite levels between
control and ΔyhfW (Fig.5). The promoter region of yhfW contains
(predicted) binding sites for regulators of the citric acid cycle and citrate
uptake CcpC and CitT (Table 5). It was therefore interesting to see
significant changes in several TCA cycle metabolites at 6 hrs, such as
fumarate, 2-oxoglutarate, and citrate. There were also significant changes
in amino acids and amino acid intermediates such as L-threonine, 2-
oxoglutarate, phenylpyruvate, L-methionine, L-tryptophan, L-aspartate
and L-glutamate. Other significant changes were found in dCTP an dTTP
as well as the cell-wall metabolite N-acetyl muramoyl-Ala. At 7 hrs fewer
differences in metabolites were found. Significantly changed were N-acetyl
muramoyl-Ala, UDP-MurNac, GDP and FAD.
Competence subpopulations
103
The changes in TCA cycle are interesting as mutaions in TCA cycle genes
can lead to defects in sporulation. (Craig et al., 1997; Fortnagel and Freese,
1968, 1968; Ireton et al., 1995). Mutations have a negative effect on
phosphorelay activation resulting in lower levels of Spo0A~P (Craig et al.,
1997; Ireton et al., 1995). The TCA cycle is linked to amino acid metabolism
and it is possible that the reduction of aminoacid levels is a result of defects
in the TCA cycle.
Transcriptomic analysis of BFA1698 (ΔyhfW)
To determine if there are changes in amino acid biosynthesis and TCA cycle
genes in the mutant we performed RNA-seq on samples harvested at the
same time in the same experiment as those used for the metabolomics
experiment. Although there are quite a few metabolites significantly
changed we only found 17 differentially expressed genes in the RNA-seq
data (Table 6). None of the genes found are known amino acid or TCA cycle
genes. Interestingly we did find up-regulation of NAD biosynthesis genes
nadA ,nadB and nadC. Biosynthesis of NAD in B. subtilis occurs from
aspartate and uses fumarate or oxygen as electron acceptor for FAD
reoxidation (Marinoni et al., 2008). The expression of the three NAD
synthesis genes is low under competence conditions in WT B. subtilis
(Nicolas et al., 2012). It is possible that the changes in TCA cycle and a
possible resulting defect in NAD/NADH homeostasis is responsible for up-
regulation of NAD synthesis genes, or the up-regulation of NAD synthesis
disrupts NAD/NADH homeostasis. Interestingly we do not see a significant
increase in the levels of NAD nor in the levels of NADP in the
metabolomics data. It is also possible that NAD is processed further in the
Nicotinate and Nicotinamide pathway. As we do not find significant
changes in the expression levels of amino acid synthesis genes it seems
likely that the reduction in the levels of amino acid synthesis intermediates
and amino acids are the result of a disruption in the TCA cycle. We also
found up-regulation of the Na+/H+ antiporter NhaC, which may be the
result of internal pH disruptions due to the lower levels of amino acids and
intermediates such as fumarate, 2-oxoglutarate, aspartate, glutamate and
citrate. NhaC has been found to be involved in pH homeostasis and the
uptake of Na+ (Prágai et al., 2001).
Chapter 3
104
Fig. 5 Significantly changed metabolites. (A, B, C). relative amounts detected by LC-MS at 6hrs. Statistics two. (D, E). Absolute amounts detected by GC-MS at 6hrs. Statistics T-test. (F). relative amounts detected by LC-MS at 7hrs. Statistics were done using a 2 tail T-test, for samples indicated by asterisk a rank-sum test was performed. Error bars respresent the standard d.eviation
Competence subpopulations
105
The majority of the down-regulated genes have no known function, but the
expression pattern of yxeD and sspD is very similar to that of yhfW
(Nicolas et al., 2012) (subtiwiki). Because the down-regulated genes have
no known function in either B. subtilis or other species further research is
required to determine what role they have in the metabololomic changes.
gene fold description
nadB 39.1 L-aspartate oxidase
nadC 35.5 nicotinate-nucleotide diphosphorylase (carboxylating)
nadA 29.2 quinolinate synthetase
lip 11.7 extracellular lipase
trnY-Phe 7.3 transfer RNA-Phe
nhaC 5.5 Na+/H
+ antiporter
tyrS 5.2 tyrosyl-tRNA synthetase
yrzI 4.3 Unkown
opuCB 4 glycine betaine/carnitine/choline ABC transporter
ykzN -3.7 Unkown
corA -6.1 Unkown
ywjC -8.7 Unkown
ywqJ -11.9 Unkown
yosF -42 Unkown
sspP -79.5 probable small acid-soluble spore protein
yxeD -204.3 Unkown
ywqI -334.7 Unkown
Effects of yhfW deletion on sporulation
Although the effect of deletion of yhfW on spo0A expression is not
statistically significant under competence conditions there is a clear
difference. As yhfW is primarily regulated by SigF, we decided to
determine whether absence of yhfW could lead to a significant difference in
spo0A expression under sporulation conditions.
Table 6. Differential gene expression of BFA1698 under competence stimulating conditions.
Chapter 3
106
BFA1698 (ΔyhfW) was grown in chemically defined sporulation medium
(CDSM) containing alanine. In contrast to the competence stimulating
conditions growth in CDSM significantly affects the expression of spo0A.
Interestingly the expression of spo0A is higher in the mutant compared to
the control, whereas the expression of spo0A was lower in the mutant
under competence stimulating conditions (Fig. 6). We also looked at spore
outgrowth. The spore crops were diluted to the same OD and heated for 10
minutes at 80°C and plated on LB agar and grown overnight at 37°C. There
is no difference in the number of colonies formed between the control and
ΔyhfW, but strikingly the mutant contains a significantly higher number of
large colonies (Fig. 7). The larger size of the colonies could indicate faster
germination or a different response to the heat treatment. Sub-lethal heat
exposure has been shown to affect germination (Smelt et al., 2008).
Different methods of spore isolation and investigations into heat resistance
of the spores should lead to more insight into the effect of YhfW on
sporulation and spore properties. Investigations into the physical spore
properties may lead more insight in the apparent increased germination as
YhfW has been found as a putative spore coat protein by (Abhyankar et al.,
2015).
High levels of citrate as a result of mutations in TCA cycle genes has been
indicated as being responsible for defects in sporulation (Craig et al., 1997;
Matsuno et al., 1999). We find under competence conditions lower levels of
citrate. As we found higher levels of spo0A expression under sporulation
conditions, it would be interesting to determine citrate levels under
sporulation conditions.
Competence subpopulations
107
Fig. 6(A). Growth of BFA1698_Pspo0A -gfp (red) and Pspo0A -gfp (control , green) in CDSM + alanine. (B). Expression of spo0A in CDSM + alanine red BFA1698 green control.There is a statistically significant difference in both the growth P<0.001 and spo0A expression P 0.0001 between BFA1698 and the control (Mann-Whitney U-test)
Chapter 3
108
Discussion
In this study we attempted to elucidate some of the remaining questions
regarding the K-state of B. subtilis. Is there differential expression of non-
coding RNAs during competence? Does ComK directly down-regulate
genes in the competent subpopulation? Are there competence proteins that
are primarily regulated at the post transcriptional level? Our results are
largely in accordance with previous studies with regard to the core ComK
regulon. Unlike previous studies, we did find six genes that were
significantly down regulated in the competent subpopulation, four of which
have a corresponding up-regulated antisense RNAs. These are degU, jag,
sigA, and lipL. The up-regulation of antisense RNAs and the presence of K-
boxes in their promoter region indicates that down-regulation of the target
genes by ComK is mostly indirect and may primarily occur through
antisense RNA. Further studies are required to confirm direct regulation of
the ncRNAs by ComK. The recently discovered Kre is so far the only gene
found that may be directly inhibited by ComK as its expression is repressed
in competent cells, and it contains several ComK binding sites (Gamba et
al., 2015). Expression of antisense RNA may be a method of fine tuning
expression.
Fig. 7. Growth on LB of colonies germinated from spores. (A). Control. (B) BFA1698 There is no significant difference in the number of colonies, however there is a significant difference in the size of the colonies P<0.001 (Mann-Whitney U-test)
Competence subpopulations
109
During competence there may be a reduced demand for SigA regulated
genes as cell division and replication are halted. DegU is a regulator of
competence as well as other processes. Down-regulation of LipL may be
related to the lack of division by competent cells. LipL is essential for lipoic
acid formation, which is necessary for the pyruvate dehydrogenase
complex of which one subunit affects Z-ring formation (Christensen et al.,
2011; Martin et al., 2011; Monahan et al., 2014). S1458 also covers pta
which has been found to affect cell-division in E. coli (Maciąg-Dorszyńska
et al., 2012). The up-regulation of their antisense RNA to these genes
during competence in B. subtilis may indicate a role in regulating cell
division during competence. Known genes affecting cell division during
competence are maf, noc and minD, unlike the gene for competence cell
division inhibitor maf; noc and minD are not differentially expressed at the
RNA level. They do however show increased protein levels in the
competent subpopulation. These results indicate that regulation of cell
division for MinD and Noc may for a large part take place on a post-
transcriptional level. Aside from the before mentioned MinD and Noc the
competence proteins SbcC and SbcD also have increased protein levels in
the competent subpopulation but are not differentially expressed at the
RNA level. Our results, combined with previous research, show that the
important competence factors MinD, Noc, SbcC and SbcD are regulated at
the post-transcriptional level.
Only five known competence genes were not significantly up-regulated
either at the RNA or protein level; these are addA, addB, hlpB, yhjB and
yhjC. This strongly indicates that their basal levels are sufficient for
competence. Differences in gene expression in the competent and non-
competent subpopulations at the two time points primarily involves amino
acid biosynthesis genes. For both the expression of amino-acid synthesis
genes is higher at the first time point. The competent sub-population may
require higher amino acid production in order to make the proteins
necessary for the rather large competence machinery. As the non-
competent sub-population enters the stationary state the demand for
protein synthesis likely decreases as the growth rate is reduced. The
decrease of Srf proteins and the increase of MecA levels at T2 are expected
as competence is a transient state requiring removal of ComK.
Chapter 3
110
One of our goals was to determine if there are genes involved in
competence that were not found in the previous transcriptomic studies. We
did indeed find up-regulation of several genes that have not been found
previously, but most notably we found strong up-regulation of yhfW,
encoding a protein with an unknown function. Deletion of yhfW leads
reduction in comG expression and causes a change in expression under
competence conditions of the important B. subtilis regulators comK, and
srfA. Its neighbouring gene yhxC which has a nearly identical expression
profile to yhxC also affects expression of comG, comK, and srfA. In
contrast to yhfW deletion of yhxC results in a strong decrease in the
number of competent cells.
YhfW and YhxC are conserved within sporulating bacteria closely related to
B. subtilis, the majority of which also contain comK, srfA and spo0A.
absence of YhfW under competence conditions results in a significant
decrease of several TCA cycle metabolites, and aminoacids. Aside from its
effect on competence deletion of yhfW significantly increases expression of
spo0A under sporulation conditions. Interestingly it may also affect the
speed of germination resulting in larger colonies. The probable increased
speed of spore outgrowth is particularly interesting with regards to the
results of (Abhyankar et al., 2015) who indicated YhfW as a putative spore
coat protein. Abhyankar et al. also found YhxC in the spore coat. The exact
effect of YhfW on germination and spore properties merits further
investigation.
YhfW deserves further investigation because absence of YhfW affects TCA
cycle components and citrate mutations in TCA cycle genes have been show
to affect sporulation (Craig et al., 1997; Fortnagel and Freese, 1968, 1968;
Ireton et al., 1995). Ireton et al. proposed that accumulation of citrate in a
citB mutant strain could be responsible for the sporulation defect, as citrate
can chelate divalent cations required for phosphorelay activity (Ireton et
al., 1995). We find lower levels of citrate during competence, but
interestingly Spo0A expression levels are lower during this condition,
although not statistically significantly lower. In contrast, there is a
statistically significant increase in Spo0A expression under sporulation
conditions and it would therefore be very interesting to determine citrate
levels under sporulation conditions.
Competence subpopulations
111
Although yhfW is confirmed to be regulated by SigF no other SigF-
regulated genes are differentially expressed between the two
subpopulations, nor is there a difference in expression of sigF. Further
investigation into the other predicted regulators is therefore of interest as it
is likely that the up-regulation under competence conditions is the result of
one or more of the other predicted regulators.
Together with our data, the presence of these genes in Bacillus species that
likely are capable of sporulation, competence and surfactin production
indicates involvement of these genes in these adaptive strategies. These
two genes are interesting because they are predicted to be enzymes and
may represent a novel way of affecting differentiation in Bacillus. In the
case of YhfW this regulation may occur through an effect on the TCA cycle.
YhfW could have a pleiotropic metabolic function that is important for
both competence and sporulation. Both competence and sporulation have
metabolic demands that are different from those during vegetative growth.
To conclude, our data confirm that ComK is primarily a transcriptional
activator and that down-regulation by ComK is indirect and occurs through
specific ncRNAs. A small number of the known competence genes, in
particular those involved in halting cell division, are primarily regulated at
the protein level rather than at the transcriptional level. The high
sensitivity of RNA-seq did indeed lead to the identification of a new and
important gene, yhfW, which together with yhxC may play an important
role in the adaptive lifestyles of Bacillus. For further research it would be
interesting to study the ncRNAs differentially expressed during
competence and confirm their regulation by ComK. Of particular interest is
the role of yhfW during sporulation, as this is when its highest expression
occurs.
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Experimental procedures
Growth conditions
Unless otherwise indicated, we used the following competence medium
adapted from (Spizizen 1958) and (Konkol et al., 2013): 18ml demi water,
2ml 10X competence medium stock ( 0.615M K2HPO4 . 3H2O, 0.385M
KH2PO4, 20% glucose, 10ml 300mM Tri-Na-citrate, 1ml 2% ferric NH4
citrate, 1g casein hydrolysate (Oxoid), 2g potassium glutamate) 100μl
2mg/ml tryptophan, 67μl 1M MgSO4 Strains were streaked out from -80
stocks on Luria Bertani (LB) agar plates with antibiotics and grown
overnight at 37°C. A single colonies (sc) was diluted 1000X in PBS or 1X
Spizizen solution 100μl of the sc colony solution was added to 20ml
medium in 100ml Erlenmeyer flasks and grown at 37°C 220rpm
Exponential/early stationary overnight cultures were diluted to an OD600
of 0.05 in 20ml medium without antibiotics. Antibiotic concentrations
chloramphenicol (cm) 5μg/ml, spectinomycin (sp) 50μg/ml, erythromicin
(ery) 0.5 μg/ml, lincomycin 12.5 μg/ml. Growth conditions in CDSM
(Hageman et al., 1984; Vasantha and Freese, 1980) + alanine
(10mM)+tryptophan 1mM. Single colonies in triplicate were grown
overnight at 37°C on LB agar + chloramphenicol (control) or
chloramphenicol + erythromycin (BFA1698) were diluted and incubated in
2ml LB 37°C 220rpm in test tubes overnight cultures were mid exponential
after overnight growth. Cultures were diluted to OD600 0.05 in 2 CDSM+
alanine +tryptophan and chloramphenicol (control) or chloramphenicol +
erythromycin (BFA1698) in test tubes and grown to mid-exponential
growth at 37°C 220rpm cultures were diluted to OD600 0.1 in 100μl
CDSM+alanine+tryptophan without antibiotics in a 96 wells plate and
grown at 37°C, 240rpm, 10min measuring interval for 20hrs in a Thermo
Fisher Varioskan Lux . The remainder of the cultures was grown for 24hrs
after which the cultures were kept in the dark at 4°C without shaking for 4
days. Spores were harvested by centrifugation at 10000g and washing 3X
with double distilled water. The spore crops were diluted to the same OD
and heated for 10 minutes at 80°C and dilutions were plated on LB agar
with chloramphenicol and grown overnight at 37°C.
Competence subpopulations
113
Growth conditions for RNA-seq and proteomics
B. subtilis 168 pcomG-gfp chloramphenicol resistant variant was created by
Jan Willem Veening. B. subtilis 168 pcomG-gfp was grown in 1X Competence
medium, adapted from (Spizizen 1958) and (Konkol et al., 2013)
containing 18ml demi water, 2ml 10X competence medium stock ( 0.615M
K2HPO4 . 3H2O, 0.385M KH2PO4, 20% glucose, 10ml 300mM Tri-Na-
citrate, 1ml 2% ferric NH4 citrate, 1g casein hydrolysate (Oxoid), 2g
potassium glutamate) 100μl 2mg/ml tryptophan, 67μl 1M MgSO4. Cultures
were grown overnight Cultures should be late exponential early stationary
after overnight growth. Samples for protein analysis and RNA-seq analysis
were taken at 5.5hrs and 6.5hrs One hour of sorting through FACS yields
approximately 3x10^7 GFP-negative (non-competent cells) and 1.5x10^7
GFP-positive (competent) cells.
Protein sample preparation and analysis
A non-sorted control of 4.106 cells was taken. A total of 4 biological
replicates were used for the protein analysis. Samples were sorted onto a
vacuum manifold filter system. Proteins were isolated and prepared for
ESI-MS details regarding the digestion and ESI-MS settings can be found
in Supplementary material S5.
Sample preparation for RNA-seq
Samples were harvested at 5.5 and 6.5hrs diluted in 2M NaCL 1XPBS and
run through BDFACS Aria at 4⁰C samples were sorted into 4M NaCl 1X
PBS. Samples were filtered using a syringe and 13mm 0.22μm filter and
washed using TE + 20mM sodium azide and put to liquid nitrogen. The
cells on the filter were homogenized in a bead mill , and RNA was extracted
as described in (Nicolas et al., 2012a). Two biological replicates were sent
for sequencing by Primbio on a proton pI chip without ribosomal RNA
depletion. Results were analysed using T-REx
(http://genome2d.molgenrug.nl) (EdgeR trimmed-median mean method
(TMM) normalization).
Chapter 3
114
Comparisons were made between competent vs non-competent cells at T1
(5.5hrs), competent vs non-competent cells at T2 (6.5hrs), competent T1 vs
competent cells T2, non-competent T1 vs non-competent cells T2. Samples
for the RNA-seq analysis of BFA1698 were harvested and extracted as
described in Nicolas et al.
Strain Construction
BFA1698 (ΔyhfW) and BFA1701(ΔyhxC) were made using pMUTIN4 by
Dr. Rob Meima. BFA1698 and BFA1701 were transformed with genomic
DNA from B.subtilis 168 Pcomg-gfp, B.subtilis 168 PcomK-gfp, B.subtilis
168 Pspo0A-gfp, B.subtilis 168 Prok-gfp, B. subtilis 168 PsrfA-gfp. Strain list
supplementary material S6
FACS analysis of regulators in BFA1698 ΔyhfW and BFA1701 ΔyhxC
background
3 single colony replicates were inoculated and grown as described under
growth conditions. Samples were analysed every hour on a BD FACSCanto
machine. Data were analysed using Flowing Software 2.5.1. Statistics were
performed in Sigma plot using a Rank Sum test.
Supplementary material is available upon request.
References
Abhyankar, W., Pandey, R., Ter Beek, A., Brul, S., de Koning, L.J., and de Koster, C.G.
(2015). Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins
during maturation. Food Microbiol. 45, Part A, 54–62.
Arrieta‐Ortiz, M.L., Hafemeister, C., Bate, A.R., Chu, T., Greenfield, A., Shuster, B., Barry,
S.N., Gallitto, M., Liu, B., Kacmarczyk, T., et al. (2015). An experimentally supported
model of the Bacillus subtilis global transcriptional regulatory network. Mol. Syst. Biol. 11.
Berka, R.M., Hahn, J., Albano, M., Draskovic, I., Persuh, M., Cui, X., Sloma, A., Widner, W.,
and Dubnau, D. (2002). Microarray analysis of the Bacillus subtilis K-state: genome-wide
expression changes dependent on ComK. Mol. Microbiol. 43, 1331–1345.
Briley Jr, K., Prepiak, P., Dias, M.J., Hahn, J., and Dubnau, D. (2011). Maf acts downstream
of ComGA to arrest cell division in competent cells of B. subtilis. Mol. Microbiol. 81, 23–
39.
Referencees
115
Brown, A.L., and Smith, D.W. (2009). Improved RNA preservation for immunolabeling and
laser microdissection. RNA 15, 2364–2374.
Christensen, Q.H., Martin, N., Mansilla, M.C., de Mendoza, D., and Cronan, J.E. (2011). A
Novel Amidotransferase Required for Lipoic Acid Cofactor Assembly in Bacillus subtilis.
Mol. Microbiol. 80, 350–363.
Craig, J.E., Ford, M.J., Blaydon, D.C., and Sonenshein, A.L. (1997). A null mutation in the
Bacillus subtilis aconitase gene causes a block in Spo0A-phosphate-dependent gene
expression. J. Bacteriol. 179, 7351–7359.
Fortnagel, P., and Freese, E. (1968). Analysis of Sporulation Mutants II. Mutants Blocked
in the Citric Acid Cycle. J. Bacteriol. 95, 1431–1438.
Gamba, P., Jonker, M.J., and Hamoen, L.W. (2015). A Novel Feedback Loop That Controls
Bimodal Expression of Genetic Competence. PLoS Genet. 11.
Hageman, J.H., Shankweiler, G.W., Wall, P.R., Franich, K., McCowan, G.W., Cauble, S.M.,
Grajeda, J., and Quinones, C. (1984). Single, chemically defined sporulation medium for
Bacillus subtilis: growth, sporulation, and extracellular protease production. J. Bacteriol.
160, 438–441.
Hahn, J., Tanner, A.W., Carabetta, V.J., Cristea, I.M., and Dubnau, D. (2015). ComGA-RelA
interaction and persistence in the Bacillus subtilis K-state. Mol. Microbiol. 97, 454–471.
Haijema, B.-J., Hahn, J., Haynes, J., and Dubnau, D. (2001). A ComGA-dependent
checkpoint limits growth during the escape from competence. Mol. Microbiol. 40, 52–64.
Hamoen, L.W., Smits, W.K., de Jong, A., Holsappel, S., and Kuipers, O.P. (2002). Improving
the predictive value of the competence transcription factor (ComK) binding site in Bacillus
subtilis using a genomic approach. Nucleic Acids Res. 30, 5517–5528.
Ireton, K., Jin, S., Grossman, A.D., and Sonenshein, A.L. (1995). Krebs cycle function is
required for activation of the Spo0A transcription factor in Bacillus subtilis. Proc. Natl.
Acad. Sci. 92, 2845–2849.
Irnov, I., Sharma, C.M., Vogel, J., and Winkler, W.C. (2010). Identification of regulatory
RNAs in Bacillus subtilis. Nucleic Acids Res. 38, 6637–6651.
de Jong, A., van der Meulen, S., Kuipers, O.P., and Kok, J. (2015). T-REx: Transcriptome
analysis webserver for RNA-seq Expression data. BMC Genomics 16, 663.
Kanamaru, K., Stephenson, S., and Perego, M. (2002). Overexpression of the PepF
oligopeptidase inhibits sporulation initiation in Bacillus subtilis. J. Bacteriol. 184, 43–50.
Konkol, M.A., Blair, K.M., and Kearns, D.B. (2013). Plasmid-Encoded ComI Inhibits
Competence in the Ancestral 3610 Strain of Bacillus subtilis. J. Bacteriol. 195, 4085–4093.
Maciąg-Dorszyńska, M., Ignatowska, M., Jannière, L., Węgrzyn, G., and Szalewska-Pałasz,
A. (2012). Mutations in central carbon metabolism genes suppress defects in nucleoid
position and cell division of replication mutants in Escherichia coli. Gene 503, 31–35.
Chapter 3
116
Marinoni, I., Nonnis, S., Monteferrante, C., Heathcote, P., Härtig, E., Böttger, L.H.,
Trautwein, A.X., Negri, A., Albertini, A.M., and Tedeschi, G. (2008). Characterization of l-
aspartate oxidase and quinolinate synthase from Bacillus subtilis. FEBS J. 275, 5090–5107
Martin, N., Christensen, Q.H., Mansilla, M.C., Cronan, J.E., and de Mendoza, D. (2011). A
Novel Two-Gene Requirement for the Octanoyltransfer Reaction of Bacillus subtilis Lipoic
Acid Biosynthesis. Mol. Microbiol. 80, 335–349.
Matsuno, K., Blais, T., Serio, A.W., Conway, T., Henkin, T.M., and Sonenshein, A.L. (1999).
Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus
subtilis. J. Bacteriol. 181, 3382–3391
Mirouze, N., Ferret, C., Yao, Z., Chastanet, A., and Carballido-López, R. (2015). MreB-
Dependent Inhibition of Cell Elongation during the Escape from Competence in Bacillus
subtilis. PLoS Genet 11, e1005299.
Monahan, L.G., Hajduk, I.V., Blaber, S.P., Charles, I.G., and Harry, E.J. (2014). Coordinating
Bacterial Cell Division with Nutrient Availability: a Role for Glycolysis. MBio 5.
Nicolas, P., Mäder, U., Dervyn, E., Rochat, T., Leduc, A., Pigeonneau, N., Bidnenko, E.,
Marchadier, E., Hoebeke, M., Aymerich, S., et al. (2012). Condition-Dependent
Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis. Science 335,
1103–1106.
Nilsson, H., Krawczyk, K.M., and Johansson., M.E. (2014). High salt buffer improves
integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells. J.
Biotechnol. 192, Part A, 62–65.
Ogura, M., and Tsukahara, K. (2010). Autoregulation of the Bacillus subtilis response
regulator gene degU is coupled with the proteolysis of DegU-P by ClpCP. Mol. Microbiol.
75, 1244–1259.
Ogura, M., Yamaguchi, H., Kobayashi, K., Ogasawara, N., Fujita, Y., and Tanaka, T. (2002).
Whole-Genome Analysis of Genes Regulated by the Bacillus subtilis Competence
Transcription Factor ComK. J. Bacteriol. 184, 2344–2351.
Prágai, Z., Eschevins, C., Bron, S., and Harwood, C.R. (2001). Bacillus subtilis NhaC, an
Na+/H+ Antiporter, Influences Expression of the phoPR Operon and Production of
Alkaline Phosphatases. J. Bacteriol. 183, 2505–2515.
Smelt, J.P.P.M., Bos, A.P., Kort, R., and Brul, S. (2008). Modelling the effect of sub(lethal)
heat treatment of Bacillus subtilis spores on germination rate and outgrowth to
exponentially growing vegetative cells. Int. J. Food Microbiol. 128, 34–40.
Smits, W.K., Eschevins, C.C., Susanna, K.A., Bron, S., Kuipers, O.P., and Hamoen, L.W.
(2005). Stripping Bacillus: ComK auto-stimulation is responsible for the bistable response
in competence development. Mol. Microbiol. 56, 604–614.
Referencees
117
Spizizen, J. (1958). Transformation of biochemically deficient strains of Bacillus subtilis by
deoxyribonucleate. Proceedings of the National Academy of Sciences, 44(10), 1072-1078.
Vasantha, N., and Freese, E. (1980). Enzyme changes during Bacillus subtilis sporulation
caused by deprivation of guanine nucleotides. J. Bacteriol. 144, 1119–1125
Wang, S.T., Setlow, B., Conlon, E.M., Lyon, J.L., Imamura, D., Sato, T., Setlow, P., Losick,
R., and Eichenberger, P. (2006). The Forespore Line of Gene Expression in Bacillus subtilis.
J. Mol. Biol. 358, 16–37.
Yoshida, K., Yamaguchi, H., Kinehara, M., Ohki, Y., Nakaura, Y., and Fujita, Y. (2003).
Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a
TnrA box. Mol. Microbiol. 49, 157–165.